Composition and methods for processing a sputum sample

ABSTRACT

The present disclosure relates to a composition and its use for treating a sputum sample suspected to contain mycobacteria. The composition comprises thymol, a linear or branched alcohol, a chaotropic agent, a reducing agent, a detergent, and a buffer, and has a pH value between 8.5 and 10. Also disclosed is a method for treating a sputum sample suspected to contain mycobacteria.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to European ApplicationNo. EP 17210184.2, filed on Dec. 22, 2017, the content of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention belongs to the field of processing biologicalsamples, such as a sample containing bacteria. Within this field, itconcerns a composition and a method for treating a sputum sample.

BACKGROUND OF THE INVENTION

The isolation of biological materials such as nucleic acids or proteinsfrom complex biological mixtures such as e.g. clinical samples has beenof considerable significance especially for diagnostic purposes.

Examples for diagnostic applications of biological sample preparationcomprise preparation and subsequent detection of viruses or bacterialtargets. Especially in a clinical environment, such techniques oftenrequire the handling of highly pathogenic material such as livingbacterial cells that may pose a significant risk of infection for theperson conducting the preparation. Therefore, it may be important toinactivate the respective pathogens as a safety measure. In manyinstances, such inactivation may be achieved by a typical preparationstep in which viral particles or bacterial cells are lysed such that therespective contents are released, before further measures for theenrichment of the analyte in question may be applied. Standard lysisprocedures for most of the common viral particles and bacterial cellsare well-established and known to the person of skill in the art.

However, certain pathogens require a more rigorous treatment for asuccessful inactivation, including species of the Mycobacteriumtuberculosis complex (MTBC), in the following also referred to as“mycobacteria”. These bacteria are enveloped by a relatively thick andcomplex cell wall that exhibits a considerably higher robustness thantheir counterparts found in most other clinically relevant bacteria.

An additional challenge is provided by the samples usually required forthe diagnosis of mycobacteria. MTBC species are typically detected insputum, which is per se a demanding sample matrix requiring more complextreatment than many other clinical sample types such as, for instance,blood plasma. Raw sputum tends to be a highly viscous matter oftenrequiring liquefying before it can be processed.

Pertinent approaches applied in the art include, for example, U.S. Pat.No. 862,782, teaching the use of compositions contain varioussurfactants, chaotropes, buffers, detergents, and other components.Practical use has shown that inactivation of mycobacteria may not alwaysbe optimal, as described herein below.

The present disclosure describes an improved approach for processingsputum samples suspected to contain mycobacteria.

SUMMARY OF THE INVENTION

In a first aspect, a composition is disclosed for treating a sputumsample suspected to contain mycobacteria. The composition provides forliquefying of the sputum which is otherwise often difficult to handle,particularly with common liquid handling systems such as pipettors. Thecomposition further allows for the inactivation of the mycobacteria ifpresent in the sample. Its active ingredients include thymol, a linearor branched alcohol, a chaotropic agent, a reducing agent, a detergent,and a buffer. The pH value of the composition is between 8.5 and 10.

Another aspect disclosed herein is a method for treating a sputum samplesuspected to contain mycobacteria. In brief, the sputum is contacted andmixed with a composition as described herein, then incubated undersuitable conditions for liquefying the sputum and inactivation of themycobacteria.

A further aspect disclosed herein is the use of a composition asdescribed herein for treating a sputum sample suspected to containmycobacteria.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect described herein is a composition treating a sputumsample suspected to contain mycobacteria, the composition comprising thefollowing components:

-   -   thymol    -   a linear or branched alcohol    -   a chaotropic agent    -   a reducing agent    -   a detergent    -   a buffer,

wherein the composition has a pH value between 8.5 and 10.

Tuberculosis (TB) is a bacterial infection caused by species of theabove-described Mycobacterium tuberculosis complex (MTBC). TB remains amajor global health problem, ranking among the top 10 causes of death,and the emergence of drug-resistant strains of M. tuberculosis is agrowing threat worldwide. The diagnosis of TB is confirmed by recoveryof M. tuberculosis bacilli from clinical samples—for pulmonarytuberculosis from respiratory samples such as sputum. As mentionedabove, those samples constitute a major challenge in terms of biosafety,further processing in automated workflows due to high sample viscosityand compatibility for diagnostic testing because of high loads of sampleinherent interfering substances. Currently, there is no commercialreagent available that adequately balances all of above mentionedchallenges. The composition described herein allows to effectivelyinactivate Mycobacterium tuberculosis complex bacilli up to 5E+07CFU/mL, liquefies respiratory samples and is compatible with automatedsample extraction workflows that can be used in molecular diagnosis witha reagent stability of equal to or greater than 18 months.

“Sputum” is the thick mucus or phlegm that is expelled from the lowerrespiratory tract (bronchi and lungs) through coughing; it is not salivaor spit. Typically, sputum includes a considerable amount of theglycoprotein sputum. In the sample collection process, the sample isretrieved from the lower airways and not from the upper respiratorytract. An unprocessed sample of sputum is also referred to herein as“raw sputum”. In some embodiments described herein, the sputum sample isa raw sputum sample. Alternatively, the sputum collected from a patientmay be diluted with, for example, a common buffer such as Tris, citrate,or the like. Such buffers do typically not substantially contribute toaltering the difficult handling properties of a sputum sample, i.e. thesputum may still remain a viscous and mostly immiscible matter withinthe diluent. In such cases, addition of a composition such as thecomposition disclosed herein will be required in order to make thesputum amenable to further processing by conventional liquid handlingdevices and methods.

The presently disclosed composition, in some embodiments an aqueoussolution, comprises various chemical compounds with distinct functionalactivities allowing for liquefaction of a patient sample (digestion ofthe sample matrix by reduction of disulfide bonds of mucin-richrespiratory samples by a reducing agent) and inactivation of M.tuberculosis bacilli by bacterial cell wall damage, protein denaturationand other mechanisms by a detergent, a chaotropic agent, a linear orbranched alcohol, and thymol as a naturally occurring biocide withstrong antimicrobial attributes. Integrity of clinically relevantbiological material, especially the mycobacterial nucleic acids, issubstantially maintained such that it can be subjected to downstreamanalysis. The pH of the reagent has a value of between 8.5 and 10 suchthat it balances between nucleic acid stability and reduced interferingcharacteristics of sample inherent substances such as mucin proteins bypH-dependent conformational changes.

“Thymol” (2-isopropyl-5-methylphenol) is a natural monoterpene phenolderivative of cymene, found in oil of thyme, and extracted from Thymusvulgaris (common thyme) and various other kinds of plants as a whitecrystalline substance. It is only slightly soluble in water at neutralpH, but highly soluble in alcohols and other organic solvents. It isalso soluble in strongly alkaline aqueous solutions due to deprotonationof the phenol moiety.

Thymol may be present in the composition described herein in a range ofdifferent concentrations. In some embodiments, it is applied in aconcentration (w/v) from 0.75%, 1%, or 1.5% to 2%, 3.5%, or 5%. In amore specific embodiment, thymol is present in a concentration of about1% (w/v). As the inventors have shown, thymol has proven very effectiveas a part of the composition disclosed herein in the context ofinactivating members of the MTBC.

The “linear or branched alcohol” also contributes to inactivation ofmycobacteria that may be present in the sputum sample, especially incombination with thymol. Among linear or branched alcohols useful in thecontext described herein are, for example, short-chain alkanols with upto 10 carbon atoms chain length. It may be a primary, secondary, ortertiary alcohol. In specific embodiments, it may be a secondaryalcohol. In more specific embodiments, the alkanol chain has from 1 to 5carbon atoms, for example, methanol, ethanol, propanol, butanol, orpentanol, or any of their branched derivatives. In even more specificembodiments, the alkanol is selected from the group of propanol and itsderivatives. In a more specific embodiment, the alkanol is isopropanol.Isopropanol (also isopropyl alcohol) is a compound with the chemicalformula C₃H₈O. It is a colorless, flammable chemical compound with astrong odor. As an isopropyl group linked to a hydroxyl group, it is thesimplest example of a secondary alcohol, where the 1 carbon atomcarrying the hydroxyl group is attached to two other carbon atoms.

The linear or branched alcohol may be present in the compositiondescribed herein in concentrations ranging from 20%, 30%, or 45% to 50%,60%, 70%, or 80% (v/v). In some embodiments, the concentration is from45% to 60%. In more specific embodiments, the concentration is 60%.

“Chaotropic agents” are substances that generally disturb the orderedstructure of water molecules in solution and non-covalent binding forcesin and between molecules. They can make several contributions to theprocedure of sample preparation. Besides, chaotropic agents contributeto the disruption of biological membranes, such as plasma membranes orthe membranes of cell organelles if present. Non-limiting examples ofchaotropic agents are guanidinium salts like guanidinium thiocyanate,guanidinium hydrochloride, guanidinium chloride or guanidiniumisothiocyanate, urea, perchlorates such as potassium perchlorate, otherthiocyanates or potassium iodide or sodium iodide. In some embodimentsdescribed herein, the chaotropic agent is a guanidinium salt. In morespecific embodiments, the chaotropic agent is guanidinium thiocyanate(GuSCN).

The chaotropic agent may be present in the composition described hereinin a concentration range from 0.5 M, 1 M, or 1.2 M to 2 M, 3.5 M, or 5M. In more specific embodiments, the concentration may range from 1.2 Mto 2 M. In a yet more specific embodiment, the concentration may beabout 1.6 M.

The “reducing agent”, as used herein, can also contribute to thedenaturation of undesired components. In particular, reducing agents, aswidely known in the art, cleave inter- and intramolecular disulfidebonds, which are especially important for the tertiary structure of manyproteins. In the context described herein, the disulfide bonds of thehigh molecular weight mucin glycoproteins, predominant components ofsputum, are broken such that the overall viscosity of the sample isdecreased. Useful in the context of the invention are reducing agentssuch as e.g. dithiothreitol (DTT), but other reducing agents known inthe art such as e.g. 2-mercaptoethanol can also be employed. In someembodiments, the reducing agent is TCEP (tris(2-carboxyethyl)phosphine).Compared to the other agents mentioned above, TCEP has the advantages ofbeing odorless, a more powerful reducing agent, an irreversible reducingagent (in the sense that TCEP does not regenerate—the end product ofTCEP-mediated disulfide cleavage is in fact two free thiols/cysteines),more hydrophilic, and more resistant to oxidation in air. It also doesnot reduce metals used in immobilized metal affinity chromatography.TCEP may be used in the composition as described herein, for instance,as a hydrochloride.

The reducing agent may be present in the composition described herein inconcentrations ranging from 1 mM, 2.5 mM, 5 mM, or 10 mM to 75 mM, 100mM 250 mM, or 500 mM. In more specific embodiments, the range is from 5mM to 75 mM. In a yet more specific embodiment, the concentration isabout 50 mM.

The “detergent” used as a component of the composition described hereinalso contributes to effects such as denaturation of plasma membrane orcell wall components, denaturation of certain proteins, and the like.Detergents useful in the context described herein include ionicdetergents such as sodium dodecyl sulfate (SDS), lithium dodecylsulfate, sodium taurodeoxycholate, sodium taurocholate, sodiumglycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzenesulfonate, N-lauroyl sarcosine, or any combination thereof. In someembodiments described herein, the detergent is a non-ionic detergent,for example, Tween-20, Nonidet P40, CHAPs, a member of the Brij series,or Triton X-100. In more specific embodiments, the detergent isTween-20. Non-ionic detergents have a hydrophilic head group that isuncharged and are preferred for their ability to break lipid-lipid andlipid-protein interactions. They have limited ability to breakprotein-protein interactions and are often referred to as non-denaturingdetergents and are used to isolate biologically active membraneproteins.

The detergent may be present in the composition described herein inconcentrations ranging from 0.01%, 0.05%, or 0.1%, to 0.5%, 1%, 2.5%,5%, or 10% (w/v or v/v). In some embodiments, the concentration rangesfrom 0.1% to 1%. In more specific embodiments, the concentration isabout 0.4%.

The “buffer” mainly provides for establishing and maintaining a certainpH value or range. Suitable buffers in the context described hereininclude, for instance, tris(hydroxymethyl)aminomethane (Tris), citrate,2-(N-morpholino)ethanesulfonic acid,N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid,1,3-bis(tris(hydroxymethyl)methylamino)propane,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),3-(N-morpholino)propanesulfonic acid (MOPS),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), bicarbonate,phosphate, or any combination thereof. In more specific embodiments, thebuffer is Tris (also known as Trizma, Trizma base, THAM, or othersynonyms).

The buffer may be present in the composition described herein in aconcentration ranging from 1 mM, 10 mM, 100 mM, or 250 mM, to 750 mM, 1M, 2 M, or 5 M. In more specific embodiments, the concentration rangemay be from 375 mM to 635 mM. In yet more specific embodiments, theconcentration is about 500 mM.

The pH value of the composition disclosed herein may range from 8.5 to10. In some embodiments, it may be about 8.5, 9, 9.5 or 10. In morespecific embodiments, it ranges from 8.7 to 9.3. In yet more specificembodiments, it is about 9.

Measures and reagents are well known in the art to adjust the pH value.As the composition described herein has an alkaline pH, bases such as,for instance, alkaline bases are among the suitable substances. In someembodiments, an alkali hydroxide is used as a base. In more specificembodiments, sodium hydroxide is used. The concentration of such a basein the composition disclosed herein may range from 1 mM, 10 mM or 100 mMto 150 mM, 250 mM, or 500 mM. In more specific embodiments, theconcentration may be about 100 mM, or about 112 mM.

In a specific embodiment, the composition described herein comprises thefollowing components:

-   -   Thymol 0.75%-5%    -   Isopropanol 45-60% (v/v)    -   GuSCN 1.2 M-2 M    -   TCEP 5 mM-75 mM    -   Tween-20 0.4%    -   Trizma 375 mM-625 mM.

Also described herein is a method for treating a sputum sample suspectedto contain mycobacteria, the method comprising the following steps:

a) contacting a sputum sample with the composition disclosed herein

b) mixing the resulting fluid

c) incubating the mixture for a period of time and under conditionssufficient for liquefying the sample and inactivating mycobacteria ifpresent in the sample.

The embodiments of the composition disclosed herein also apply to themethod described herein.

The benefits of using the composition disclosed herein for treating asputum sample, especially in terms of sputum liquefaction andmycobacteria inactivation, are described elsewhere in the presentdisclosure.

The method described herein is especially useful in cases where thesputum sample is raw sputum, as detailed in the context of thecomposition disclosed herein. By means of the method and compositiondescribed herein, even raw sputum may be successfully processed withoutprior dilution. However, in case such dilution is performed, the methoddescribed herein is also applicable to diluted or otherwise pretreatedsputum.

Contacting the sputum sample with the composition disclosed herein andmixing it therewith may be performed within the same step. For instance,a vessel containing the sputum may be placed on a shaker such thatagitation of the sample may readily be performed when the composition isadded. Alternatively, sputum may be added to a vessel containing thecomposition while said vessel is vortexed or otherwise agitated. Such aworkflow may streamline the method by saving time and potentially space.Hence, in some embodiments of the method disclosed herein, step a) andstep b) are performed at the same time.

However, in other embodiments of the method, the sputum sample and thecomposition may be contacted with each other first, and then transferredto, for instance, a shaker, a stirrer, or the like, and mixedsubsequently. Therefore, in some embodiments of the method describedherein, step a) and step b) are temporally and/or locally separated.

Means for performing the mixing are known to the person of skill in theart. As mentioned above, shakers or vortexes are among the most commondevices for this purpose. In some embodiments, the mixture is shaken orvortexed for between 5 and 60 seconds. In some embodiments, this periodof time is about 30 seconds.

Incubation of the sample/composition mixture can be conducted under avariety of conditions and for different amounts of time.

In some embodiments, incubation in step c) is carried out essentially atroom temperature, abolishing the need for thermostatic equipment. Roomtemperature is usually defined in the art as a range of 20° C. to 25° C.In some embodiments, the incubation temperature is about 25° C.

Also in some embodiments, the period of time for the incubation in stepc) is at least 30 min. In some embodiments, it is between 30 and 90 min.In more specific embodiments, it is about 50 min, in other embodimentsabout 60 min.

The incubation may be combined or complemented with additional measures,such as sonication. The application of ultrasound to the mixture cancontribute to both effects mentioned above, meaning liquefaction of thesputum sample and inactivation of the mycobacteria if present in thesample. Suitable sonicators are commercially available and known to theperson skilled in the art. They may be conventional multi-purposesonicators or tailor-made for the purpose described herein.

Hence, in some embodiments of the method disclosed herein, step c)involves sonication of the mixture, either during or subsequent to theincubation.

In some embodiments, the method described herein further comprises afterstep c) one or more of the following steps:

d) isolating a biological target material from the inactivatedmycobacteria if present in the sample

e) analyzing the biological target material.

In the context of the present disclosure, the terms “isolation”,“purification” or “extraction” of a biological target material relate tothe following: Before biological target materials like, for instance,nucleic acids may be analyzed in a diagnostic assay by amplification orthe like, they typically have to be purified, isolated or extracted frombiological samples containing complex mixtures of different components.In the context described herein, the mycobacteria, if present in thesputum sample, contain a variety of different biomolecules, and in manycases only a subgroup of these molecules are of interest for a giventype of analysis. For instance, nucleic acids to be analyzed by PCR in adownstream process may need to be separated from the other biomoleculespresent in the mixture subjected to step d) of the method disclosedherein. Suitable methods for isolation are known to the person skilledin the art.

Typically, one of the first steps comprises releasing the contents ofcells or viral particles, for example, by using enzymes and/or chemicalreagents. This process is commonly referred to as lysis. For enrichmentof the analyte in question in the lysate, one useful procedure forbinding nucleic acids entails the selective binding of nucleic acids toglass or silica surfaces of binding particles such as magnetic particlesin chaotropic salt solutions and separating the nucleic acids fromcontaminants such as agarose, proteins or cell debris. The compositionused herein may be especially useful when nucleic acids are bound toglass surfaces, as the composition already contains a chaotropic agentwhich is usually necessary for such binding.

Downstream analysis of the biological target material, as performed instep e) of the method disclosed herein, may include Polymerase ChainReaction (PCR) or sequencing of nucleic acids, or antibody-mediatedassays for proteins such as ELIZA, or the like. In some embodiments ofthe method described herein, the analysis comprises qualitative and/orquantitative detection of nucleic acids by amplification. In someembodiments, the amplification technique is PCR. Other amplificationtechniques, such as isothermal amplification (LAMP, TMA, and the like),LCR, etc. may be applied for analyzing a target nucleic acid.

In accordance with the composition and the method using it as describedherein, another aspect of the present disclosure is the use of thecomposition described herein for treating a sputum sample suspected tocontain mycobacteria. If safe handling and using liquid handling devicesare desired, the use is for liquefying a sputum sample and inactivatingmycobacteria if present in the sample.

The embodiments of the composition and the method disclosed herein alsoapply to the use described herein.

EXEMPLARY EMBODIMENTS

The following Examples are meant to illustrate specific embodiments ofthe composition, method and use disclosed herein, while they are notlimiting.

Example 1 Liquid Handling

Particularly raw sputum, but often also diluted sputum, are matricesdifficult to handle and not easily pipettable.

However, upon addition of the composition disclosed herein resulting ina 1:2 ratio, the sputum gets digested and the sample can be easilypipetted afterwards.

In this example, the composition was as follows: 1.6 M GuSCN, 0.4%Tween-20, 50 mM Tris(2-carboxyethyl)phosphine (TCEP) HCl, 500 mM TrizmaBase (Tris), 112 mM NaOH, 60% 2-Isopropanol, 1% Thymol, pH 9.

Example 2 Inactivation of Two Different MTB Strains at VariousConcentration Levels (2E+06 to 5E+07 CFU/mL) was Demonstrated at ThreeDifferent Testing Sites

Experimental Setup:

Two different quantified MTB cultures (wildtype MTB and the laboratorystrain H37v) were diluted to the intended target concentrations of 5E+07CFU/mL, 1E+07 CFU/mL and 2E+06 CFU/mL. Aliquots of 0.5 mL of thecultures were mixed with 1 mL of the composition described herein (1.6MGuSCN, 0.4% Tween-20, 50 mM Tris(2-carboxyethyl)phosphine (TCEP) HCl,500 mM Trizma Base (Tris), 112 mM NaOH, 60% 2-Isopropanol, 1% Thymol, pH9). After an incubation time of 60 minutes at room temperature, thesolution was centrifuged at 3000 g for 15 minutes, the supernatant wasdiscarded and the pellet washed 2 times with DPBS (Dulbecco'sphosphate-buffered saline), before it was suspended in 0.5 mL DPBS. Forcultivation in the commercially available MGIT system (Beckton Dickinson(BD)), the solution was transferred into a MGIT tube together with 0.8mL of the BD growth media in order to monitor growth. For the solidcultures, the solution was distributed on Löwenstein-Jensen platesstored in an incubator. For the MGIT system, growth is detected based onoxygen reduction, growth is detected on solid cultures by colonyformation.

This study was conducted with two unique batches of the compositiondescribed herein, untreated samples for each concentration level wereincluded as growth controls:

-   -   Multiple replicates were treated with either batch 1 or batch 2        of the composition disclosed herein at T0, after incubation for        the intended time period and temperature, cells were washed with        PBS (phosphate-buffered saline) and growth was initiated either        according to the procedures used for the MGIT system or by        established procedures on solid media types    -   As growth controls, two replicates were treated with PBS only to        mimic the procedure

A result overview is given in the Table 1.

TABLE 1 Time point of growth detection after treatment Concentration MTB[cells/mL] 2.0E+06 1.0E+07 5.0E+07 Composition batch 1 2 n/a* 1 2 n/a* 12 n/a* MTB Site 1 no no 18-20 days no no 15-16 days no no 12-14 daysgrowth growth growth growth growth growth Site 2 no no 16 days no no 11days no no 9-10 days growth growth growth growth growth growth Site 3 nono growth no no growth no no growth growth growth detected** growthgrowth detected** growth growth detected** MTB Site 1 no no 18-21 daysno no 12-13 days no no 11-12 days H37 growth growth growth growth growthgrowth Site 2 no no 14 days no no 14-18 days no no 10-11 days growthgrowth growth growth growth growth Site 3 no no growth no no growth nono growth growth growth detected** growth growth detected** growthgrowth detected** *growth control **solid media was used (manual check),read out is not as precise as with the automated detection by MGIT

Over the time course of the study (52 days), no growth was detected inthe replicates treated with the composition disclosed herein, while allgrowth controls had detectable growth independent of the concentration.

Example 3 Effect of pH Value on Liquefaction

Mucin is a glycoprotein family abundant in sputum samples and known tobe inhibitory for, as an example, automated nucleic acid extraction.These inhibitory effects can be influenced by the protein conformationwhich is different depending on the pH value. In order to determine theoptimal pH variants of the composition described herein, different pHlevels were tested: pH 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0.

Experimental Setup:

In order to test extraction and amplification of MTB complex DNA underinhibitory conditions, 500 CFU/mL of Mycobacterium bovis BacillusCalmette-Guérin (BCG) were spiked into a matrix containing 1% mucin andtreated with one of the different pH variants of the compositiondescribed herein. Performance was evaluated by testing 5 replicates onthe Cobas® 6800/8800 systems (Roche Diagnostics) with the Cobas® MTBtest (Roche Diagnostics) according to the manufacturer's instructions.

Results are displayed in Table 2:

Channel 2 Channel 5 Positive Positive Ct [2] RFI [2] Ct [5] RFI [5] pHMean SD Mean SD Mean SD Mean SD 7.5 35.3 0.29 2.2 0.11 39.2 1.10 8.70.41 8.0 35.0 0.47 2.9 0.30 38.2 0.42 9.2 0.33 8.5 32.0 0.26 7.6 0.8731.4 0.24 9.4 0.05 9.0 31.7 0.15 9.2 0.21 30.6 0.09 9.2 0.05 9.5 32.00.22 8.9 0.23 30.8 0.19 9.2 0.05 10.0 31.5 0.17 9.4 0.20 30.6 0.12 9.30.06

The performance of any solution with a pH≥8.5 and ≤10.0 is suitable, asindicated by comparable Cts and RFIs for the target channel, as well asthe channel of the internal control.

Example 4 Optimal Concentration of Components

The tolerance ranges of concentrations of individual components of thecomposition disclosed herein were determined. In order to determinethose ranges, different variants of the solution were generated, whichwere tested against two reference batches.

-   -   a) Four components were tested due to potentially impacting the        stability and detection of nucleic acids, as well as the level        of interference caused by proteins present in the matrix:        guanidinium thiocyanate (GuSCN), Trizma Base (Tris), NaOH, and        the resulting pH value. In consequence, the following        compositions were tested:

Reference Component conc Solution 1 Solution 2 Solution 3 Solution 4GuSCN 1.6M 1.2M 1.2M 2.0M 2.0M Tween-20 0.4% (w/v) TCEP HCl 50 mMIsopropanol 60% (v/v) NaOH 112 mM Titrated for pH Trizma Base 500 mM 375mM 625 mM 375 mM 625 mM Thymol  0% (w/v) pH 9.0 8.7 9.3 8.7 9.3

Experimental Setup:

A clinical sediment panel low positive for MTB was mixed 1:9 with one ofthe compositions described herein (denoted as “solutions”, see below) orone of the two reference solutions. The respective diluted sample wasvortexed for 30 sec, incubated for 50 minutes at room temperature,sonicated and centrifuged for 1 min at 3000 g, before the sample wasloaded onto the Cobas® 6800/8800 system.

For all conditions tested all tested replicates were positive, neitherRFI nor Ct had shown any significant difference, therefore theperformance was considered equivalent.

-   -   b) Three components were tested due to potentially impacting        inactivation properties of the composition described herein:        guanidinium thiocyanate (GuSCN), isopropanol, and thymol. The        following compositions were tested:

Reference Solution Solution Solution Solution Solution Component conc 56 7 8 9 GuSCN 1.6M 1.2M 1.6M 1.6M 1.2M 1.6M Tween-20 0.4% (w/v) TCEP HCl 50 mM Isopropanol 60% (v/v) 60% (v/v) 45% (v/v)  60% (v/v)  45% (v/v)60% (v/v) NaOH 112 mM Trizma Base 500 mM Thymol  1% (w/v)  1% (w/v)  1%(w/v) 0.75% (w/v) 0.75% (w/v)  5% (w/v) pH 9.0

Experimental Setup:

Two different quantified MTB cultures (wildtype MTB and the laboratorystrain H37v) were diluted to the intended target concentrations of 5E+07CFU/mL and 2E+06 CFU/mL. Aliquots of 0.5 mL of the cultures were mixedwith either of the solutions or the reference solution. After anincubation time of 50 minutes at room temperature, the mixture wascentrifuged at 3000 g for 15 minutes, the supernatant was discarded andthe pellet washed 2 times with DPBS, before it was suspended in 0.5 mLDPBS. For cultivation in the MGIT system (BD), the solution wastransferred into a MGIT tube together with 0.8 mL of the BD growth mediain order to monitor growth. This study was conducted with two uniquebatches of the composition described herein and 5 of its variants,untreated samples for each concentration level were included as growthcontrols:

-   -   Multiple replicates were treated with either batch of the        composition at T0. After incubation for the intended time period        and temperature, cells were washed with PBS and growth was        initiated either according to the procedures used for the BD        MGIT system or by established procedures on solid media types.    -   As growth controls, two replicates were treated with PBS only to        mimic the procedure.

A result overview is given in Table 3.

TABLE 3 Time point of growth detection after treatment Concentration MTB[cells/mL] Reference Solution Solution Solution Solution Solution Strainconc 5 6 7 8 9 n/a* 2.0E+06 MTB no growth no growth no growth no growthno growth no growth 17 days MTB H37 no growth no growth no growth nogrowth no growth no growth 6-13 days 5.0E+07 MTB no growth no growth nogrowth no growth no growth no growth 6 days MTB H37 no growth no growthno growth no growth no growth no growth 3 days *growth control

Over the time course of the study (52 days), no growth was detected inthe MIS or MIS variants treated replicates, while all growth controlshad detectable growth independent of the concentration, thereforeinactivation potential is considered equal for all tested formulations.

-   -   c) TCEP, a reducing agent with major contribution to liquefying        the sputum samples, was tested in seven additional solutions in        which only the TCEP concentration was titrated:        -   0 mM TCEP        -   2.5 mm TCEP        -   5 mM TCEP        -   10 mM TCEP        -   20 mM TCEP        -   30 mM TCEP        -   40 mM TCEP        -   75 mM TCEP        -   100 mM TCEP

All other components of the composition described herein remained equalto the used reference (1.6 M GuSCN, 0.4% Tween-20, 50 mMTris(2-carboxyethyl)phosphine (TCEP) HCl, 500 mM Trizma Base, 112 mMNaOH, 60% 2-Isopropanol, 1% Thymol, pH 9)

Experimental Setup:

A 12% mucin solution, used to simulate conditions found in raw sputum,was mixed with one of the different variants of the compositiondisclosed herein, vortexed for 30 sec and incubated for 50 min at roomtemperature. Liquefaction capacities were assessed after incubation.Already 5 mM TCEP were sufficient in order to show a good liquefactionof the sample.

The entire titration study demonstrated that the following concentrationranges of the used components led to good liquefaction:

-   -   GuSCN 1.2 M-2.0 M    -   Tween-20 0.4% (w/v)    -   TCEP HCl 5-75 mM    -   Isopropanol 45-60% (v/v)    -   NaOH 112 mM    -   Trizma Base 375-625 mM    -   Thymol 0.75-5%    -   pH 8.7-9.3

Example 5 Inactivation Comparison Against Existing Products wasPerformed Directly Against the Prime Store Solution CommerciallyAvailable by the Company LongHorn

Experimental Setup:

Two different quantified MTB cultures (wildtype MTB and the laboratorystrain H37v) were diluted to the intended target concentrations of 5E+07CFU/mL and 2E+06 CFU/mL. Aliquots of 0.4 mL of the cultures were mixedwith either 0.8 mL of the composition disclosed herein or the Primestore solution. After an incubation time of 50 minutes at roomtemperature, the solution was centrifuged at 3000 g for 15 minutes, thesupernatant was discarded and the pellet washed 2 times with DPBS,before it was suspended in 0.5 mL DPBS. For cultivation in the MGITsystem (BD), the solution was transferred into a MGIT tube together with0.8 mL of the BD growth media in order to monitor growth. This study wasconducted with two unique batches of the composition described herein,untreated samples for each concentration level were included as growthcontrols:

-   -   Multiple replicates were treated with either the composition        described herein or Prime Store at T0. After incubation for the        intended time period and temperature, cells were washed with PBS        and growth was initiated either according to the procedures used        for the BD MGIT system or by established procedures on solid        media types    -   As growth controls, two replicates were treated with PBS only to        mimic the procedure

A result overview is given in Table 4.

TABLE 4 Time point of growth detection after treatment Concentration MTB[cells/ mL] 2.0E+06 5.0E+07 Strain Composition Prime Store n/a*Composition Prime Store n/a* MTB no growth 13-14 days 17 days no growth9-10 days 6 days MTB no growth 11 days 6-13 days no growth 7-8 days 3days H37 *growth control

Over the time course of the study (52 days), no growth was detected inthe replicates treated with the composition disclosed herein, while allgrowth controls and the Prime Store treated replicates had detectablegrowth independent of the concentration, therefore inactivationperformance can be considered superior in comparison to existingproducts.

The invention claimed is:
 1. A composition comprising an aqueoussolution for liquefaction of a sputum sample suspected to containmycobacteria and for inactivation of said mycobacteria, the compositioncomprising: a) thymol in a concentration from 0.75% to 5%; b)isopropanol; c) a chaotropic agent comprising guanidinium thiocyanate(GuSCN); d) a reducing agent in a concentration from 5 mM to 75 mM andselected from the group consisting of dithiothreitol (DTT),2-mercaptoethanol, and TCEP (tris(2-carboxyethyl)phosphine); e) anon-ionic detergent in a concentration from 0.1% to 1% and selected fromthe group consisting of polyoxyethylene (20) sorbitan monolaurate(Tween® 20), octylphenoxypolyethoxyethanol (Nonidet® P40),3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPs), amember of the polyoxyethylene alkyl ethers (Brij®) series, and2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton® X-100); and f)a buffer selected from the group consisting oftris(hydroxymethyl)aminomethane (Tris/Trizma®),2-(N-morpholino)ethanesulfonic acid,N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid,1,3-bis(tris(hydroxymethyl)methylamino)propane,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),3-(N-morpholino)propanesulfonic acid (MOPS), andpiperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES); wherein thecomposition has a pH value between 8.5 and
 10. 2. The composition ofclaim 1, wherein the reducing agent is TCEP(tris(2-carboxyethyl)phosphine).
 3. The composition of claim 1, whereinthe non-ionic detergent is Tween®
 20. 4. The composition of claim 1,wherein the buffer is tris(hydroxymethyl)aminomethane (Tris/Trizma®). 5.The composition of claim 1, comprising the following components: Thymol0.75%-5%; Isopropanol 45-60% (v/v); GuSCN 1.2 M 2 M; TCEP 5 mM 75 mM;Tween® 20 0.4%; and Tris/Trizma® 375 mM-625 mM.